Using the BCA assay, we determined that we had obtained 118.316 ug/mL of SDF and 1.754 ug/mL of sRAGE., We then decided to use gel electrophoresiswith transcribed RNA to determine if using different plasmids or restriction enzymes would increase the yield ofsRAGE. sRAGE yielded depended on the plasmid and not the enzyme. Bacterial colonies 5 and 10, which were transfected with Plasmid 1, providedthe highest yield and were thereby used to produce sRAGE.

The high glucose environment impairs the ability of the HL-60 cells and white blood cells to migrate towards SDF-1, ultimately impairing cell migration. The addition of sRAGE reverses the SDF-1 impairment. Current challenges include the inability to produce a high yield of sRAGE protein. This could possibly be because of the condition in which the sRAGE bacteria is grown. Changing the temperature or the pH of the media in which sRAGE is being grown could possibly increase the yield of sRAGE. Thereby, future work includes adjusting these conditions of the procedure to increase expression and yield of sRAGE. Adding additional ELP repeats onto the fusion protein proposes the possibility of lowering the transition temperature making it close to body temperature. Furthermore, future direction also includes designing a nanoparticle that contains both sRAGE and SDF-1. Because sRAGE potentiates SDF-1 activity, packaging them both in one nanoparticle would increase the efficiency of our design and decrease wound closing time. Future directions also include in vivo animal studies to analyze the wound healing kinetics of sRAGE in various conditions. From this, the stability of sRAGE nanoparticle in wound fluid will be measured and realistically compared to existing solutions.

We would like to acknowledge our mentor Dr. Berthiaume who has been involved in our project every step of the way and provided the moral support to push through the most difficult times. We would also like to thank our graduate student advisor Hwan June Kang who has guided us through all the cell studies. Thank you to Dr. Cohen who helped us with all the protein work and our student mentor Sagar Nisraiyya for guiding us through the different experiments our first semester. We would also like to thank Agnes Yeboaha for being around to answer any of our impromptu questions and provide any supplemental materials we needed. Thank you to the National Institute of Health for funding this senior design project.

Diabetic individuals have a decreased wound healing ability that causes them to suffer from chronic wounds such as foot ulcers. An important signaling pathway, SDF-1, that contributes to cell migration and cell proliferation is downregulated in a diabetic individuals. There is an increased production of advanced glycated end-products (AGEs). AGEs have a receptor RAGE and when AGE-RAGE binding occurs, there is an increase inflammation and infection at the wound site. To interfere with the AGE-RAGE binding and restore the SDF-1 pathway, sRAGE, the soluble isoform of RAGE is used. It competes with RAGE to bind to AGE instead and when the SDF-1 pathway recovers, cell migration and cell proliferation recovers to heal the wound at a normal rate.

In the United States alone, 29 million are diagnosed with diabetes and of the 15% of people who develop foot ulcers, 20% of them are non-healing.This amounts to over 870,000 patients with chronic foot ulcers. These chronic wounds take over $30,000 to treat and many have to face amputation as their only option. Current solutions include hyperbaric chambers, vacuum-assisted wound closure devices however these require multiple visits to a physician’s office and can be painful. Current wound dressings serve as a microbial and moisturizing agent but do not alter the healing process. A dressing that contains an active that will enhance the cellular process behind wound healing is very needed.

In order to produce the recombinant sRAGE protein, we took advantage of the ELP motif we attached to the protein. ELP causes the protein to self-assemble into nanoparticles below a certain transition temperature. Above this temperature, the proteins will be in solution. We transfected bacteria with a plasmid containing the sRAGE-ELP DNA. After allowing the bacteria to grow, we centrifuged and lysed them. We then took this lysate and used temperature inversion purification, as described in the figure, to purify sRAGE-ELP. To determine the amount of purified protein, we used a BCA assay and then measured the absorbance of our samples at 595nm to determine the concentration of protein in our samples. We evaluated the efficacy of the produced sRAGE protein using a transwell migration assay. HL60 cells, which were used as model responder cells, were incubated in environments of varying glucose concentrations for one day. We used a 5mM glucose environment to represent non-diabetic conditions, while a 50mM glucose environment simulated highly diabetic conditions. Cells were then transferred to the migration wells, and they were given either no bioactive molecules, SDF-1 alone, or both SDF-1 and sRAGE. After 70 minutes, we counted the number of cells that had migrated out of each of the wells

In order to test the efficacy of the produced sRAGE, we performed transwell migration assays. The control condition, which contained 5 mM glucose and no bioactive proteins, showed virtually no cellmigration (only 4.6875%). Wells that contained 5mM glucose with SDF exhibited an increased cell migration rate of 26.875%, proving that SDF does indeed lead to high cellular migration. The high glucose condition (50 mM) with SDF showed a slight decline in cellular migration to 19.165%. The final experimental diabetic condition, which contained 50mM glucose with a combination of SDF and sRAGE, displayed an increased cellular migration rate of 22.9%.

Melissa Ann Olekson’s Dissertation: STRATEGIES FOR IMPROVING GROWTH FACTOR FUNCTION IN DIABETIC WOUNDS

Faulknor, R.A., Mesenchymal stromal cells in alginate dressings to enhance chronic wound healing. 2015, Rutgers University-Graduate School-New Brunswick.

Oliveira, M.I.A., et al., RAGE receptor and its soluble isoforms in diabetes mellitus complications. Jornal Brasileiro de Patologia e Medicina Laboratorial, 2013. 49(2): p. 97-108.

Schmidt, Ann Marie, et al. "The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses." Journal of Clinical Investigation 108.7 (2001): 949

Figure: (Above) Diagram depicting the packaging of our nanoparticle. Once our nanoparticle is developed, we will experiment with different polymers and pore size, and package the nanoparticle in a combination that provides for the most mobility.